The Mixed Lineage Leukemia (MLL) gene is involved in chromosomal translocations which generate a fusion protein causing aggressive, acute leukemia with poor prognosis. The resulting chimeric protein retains the amino-terminal portion of MLL including the CXXC DNA-binding domain while the carboxy-terminal portion of the oncoprotein is comprised of the fusion partner. The CXXC domain is found within a larger repression domain and binds to non- methylated CpG DNA. This CXXC DNA-binding domain contains a specific clustering of eight cysteines which coordinate two zinc ions and is essential for the transformation capacity of cells expressing an MLL fusion protein. The closest homolog of MLL, MLL2 (alternatively named MLL4), contains a similar CXXC domain but binds with lower DNA-binding affinity. Interestingly, artificial MLL2-ENL does not transform hematopoietic progenitor cells in vitro. Amino acid differences between the CXXC domain or the larger repression domain of MLL and MLL2 must contribute to these functional differences. Specific amino acids along the DNA contact interface may play a role in differential DNA binding affinity. In addition, we have previously shown that the MLL repression domain (containing the CXXC domain) binds to co-repressor proteins. Differential binding of these repressor proteins to MLL and MLL2 may affect overall function, but might also contribute to CXXC DNA-binding affinity. Cysteine 1188, the only non-zinc-coordinating cysteine residue within the CXXC domain, is critically positioned on the DNA-binding surface and adjacent to one of the conserved cysteines involved in zinc coordination. Mutation of Cys1188 to aspartate completely abrogated the ability of the CXXC domain to bind DNA and prevented both in vitro immortalization and development of leukemia in mice when introduced in the context of MLL-AF9. In contrast, mutation of Cys1188 to alanine retained MLL's ability to bind CpG DNA, as well as to immortalize in vitro and in vivo in the context of the MLL-AF9 fusion. With a critical location on the DNA-binding surface of the CXXC domain and a thiol group susceptible to modification, we hypothesize that the side chain of Cys1188 may be physiologically altered to regulate DNA-binding affinity, allowing this residue to function as a molecular switch that regulates the binding of MLL to its non-methylated CpG DNA targets. Two specific aims are proposed in this application which will explore how specific amino acid residues of the MLL and MLL2 CXXC domains function to alter DNA binding, co-repressor binding, and susceptibility to post-translational modification. We hypothesize that these are critical determination features for MLL's role in acute leukemia. Keywords: MLL, MLL2, leukemia, hematopoiesis PUBLIC HEALTH RELEVANCE: The Mixed Lineage Leukemia (MLL) gene is involved in chromosomal rearrangements that produce MLL fusion proteins and ultimately cause acute leukemia with poor prognosis. It is not yet fully understood how the functions of MLL fusion proteins cause MLL-associated leukemia. The research proposed in this application aims to understand how the MLL functions of DNA binding and repressor protein binding are regulated to ultimately allow for therapeutic targeting of MLL fusion proteins and better clinical outcomes for this devastating form of acute leukemia.